Project summary Super-resolution optical microscopy promises to revolutionize biological imaging, as it enables non- invasive interrogation at molecular scale. Indeed, the emergence of super-resolution fluorescence microscopy has quickly impacted the way biologists study cells and subcellular phenomenon. However, super-resolution fluorescence microscopy has fundamental limitations due to the use the fluorescence as contrast mechanism. In particular, it has three major limitations: (1) it cannot reveal chemical composition of the sample; (2) it cannot interrogate small biomolecules due to the relatively bulky fluorescent tags; (3) it cannot image a large number of targets due to the color barrier (only 2~5 fluorescent colors can be practically resolved). The goal of this project is to develop a novel super-resolution imaging platform by exploiting stimulated Raman scattering (SRS) as the contrast mechanism. During the past 10 years since its invention in 2008, SRS microscopy has made widespread impact in biomedical imaging. Being a chemically sensitive method, SRS is well known for its label-free chemical analysis in a quantitative manner. With the recent development of tiny bio-orthogonal tags such as alkynes, SRS has been proven successful in interrogating a wide spectrum of small biomolecules such as lipids, glucose, amino acids, and drugs. Very recently, novel vibrational dye palettes with fine spectral resolution have been reported to achieve super-multiplex electronic pre-resonance (epr) SRS imaging of more than 20 targets simultaneously. Importantly, all these utilizes of SRS microscopy is limited by light diffraction. With SRS being a perfectly complementary contrast mechanism to the prevalent fluorescence, the current proposal aims to develop the necessary methods to bring SRS microscopy to the realm of super resolution. (1) How to improve the resolution for general chemical imaging and small biomolecule imaging; (2) how to break the diffraction limit of the super-multiplex epr-SRS imaging; (3) how to develop the matching vibrational dyes for single molecule SRS. Towards these goals, we had laid out a systematic plan as to how to crystallize this concept into a powerful technology platform. An inter-disciplinary approach has been planned out including instrumentation development, computational imaging, and novel probes synthesis. In Aim 1, we will develop and build new instrumentation. In Aim 2, we will explore new computational algorithm. In Aim 3, we will design next- generation vibrational probes. If successfully implemented, we will establish a transformative platform. The resulting super-resolution chemical imaging would find wide applications in systematically unraveling complex biological systems such as neuroscience, immunology and cancer biology for basic research, disease diagnostics and precision medicine. 1